1. Field of the Invention
The present invention relates to chimeric lyssavirus nucleic acids, and chimeric polypeptides and proteins encoded by these nucleic acids. More particularly, the invention relates to chimeric lyssavirus nucleic acids and proteins that can be used in immunogenic compositions, such as vaccines. Thus, the invention also relates to carrier molecules for expressing chimeric lyssavirus nucleic acids, methods of producing chimeric lyssavirus proteins and polypeptides, and methods of treating individuals to ameliorate, cure, or protect against lyssavirus infection. The compositions of the invention can also be used to express peptides, polypeptides, or proteins from organisms other than lyssaviruses. Thus, the invention provides methods of treating individuals to ameliorate, cure, or protect against many different infections, diseases, and disorders.
2. Background of the Related Art
Rabies is an encephalopathic disease caused by members of the Lyssavirus genus within the Rhabdoviridae family. Rabies infects all warm-blooded animals and is almost invariably fatal in humans if not treated. On the basis of nucleotide sequence comparisons and phylogenetic analyses, the Lyssavirus genus has been divided into 7 genotypes (GT). GT1 includes the classical rabies viruses and vaccine strains, whereas GT2 to GT7 correspond to rabies-related viruses including Lagos bat virus (GT2); Mokola virus (GT3); Duvenhage virus (GT4); European bat lyssavirus 1 (EBL-1: GT5); European bat lyssavirus 2 (EBL-2: GT6); and Australian bat lyssavirus (GT7).
Based on antigenicity, the Lyssavirus genus was first divided into four serotypes. More recently, this genus was divided into two principal groups according to the cross-reactivity of virus neutralizing antibody (VNAb): Group 1 consists of GT1, GT4, GT5, GT6, and GT7, while Group 2 consists of GT2 and GT3. Viruses of group 2 are not pathogenic when injected peripherally in mice. Virulence of lyssaviruses is dependent, at least in part, on the glycoprotein present in the viral coat. Interestingly, the glycoproteins of group 2 viruses show a high degree of identity, in the region containing amino acids that play a key role in pathogenicity, to the corresponding sequence of avirulent GT1 viruses (see, for example, Coulon et al., 1998, xe2x80x9cAn avirulent mutant of rabies virus is unable to infect motoneurons in vivo and in vitroxe2x80x9d, J. Virol. 72:273-278).
Rabies virus glycoprotein (G) is composed of a cytoplasmic domain, a transmembrane domain, and an ectodomain. The glycoprotein is a trimer, with the ectodomains exposed at the virus surface. The ectodomain is involved in the induction of both VNAb production and protection after vaccination, both pre- and post-exposure to the virus. Therefore, much attention has been focused on G in the development of rabies subunit vaccines. Structurally, G contains three regions, the amino-terminal (N-terminal) region, a xe2x80x9chingexe2x80x9d or xe2x80x9clinkerxe2x80x9d region, and the carboxy-terminal (C-terminal) region. (See FIG. 1.)
As depicted in FIG. 1, it is generally thought that the glycoprotein (G) ectodomain has two major antigenic sites, site II and site III, which are recognized by about 72.5% (site II) and 24% (site III) of neutralizing monoclonal antibodies (MAb), respectively. The site II is located in the N-terminal half of the protein and the site III is located in the C-terminal half of the protein. The two halves are separated by a flexible hinge around the linear region (amino acid 253 to 257).
The G ectodomain further contains one minor site (site a), and several epitopes recognized by single MAbs (I: amino acid residue 231 is part of the epitope; V: residue 294 is part of the epitope, and VI: residue 264 is part of the epitope) (Benmansour et al., 1991, xe2x80x9cAntigenicity of rabies virus glycoproteinxe2x80x9d, J. Virol. 65:4198-4203; Dietzschold et al., 1990, xe2x80x9cStructural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccinexe2x80x9d, J. Virol. 64:3804-3809; Lafay et al., 1996, xe2x80x9cImmunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sitesxe2x80x9d, J. Gen. Virol. 77:339-346; Lafon et al., 1983, xe2x80x9cAntigenic sites on the CVS rabies virus glycoprotein: analysis with monoclonal antibodiesxe2x80x9d, J. Gen. Virol. 64:843-845). Site II is conformational and discontinuous (amino acid residues 34 to 42 and amino acid residues 198 to 200, which are associated by disulfide bridges), whereas site III is conformational and continuous (residues 330 to 338). Lysine 330 and arginine 333 in site III play a key role in neurovirulence and may be involved in the recognition of neuronal receptors (see, for example, Coulon et al., supra, and Tuffereau et al., 1998, xe2x80x9cNeuronal cell surface molecules mediate specific binding to rabies virus glycoprotein expressed by a recombinant baculovirus on the surfaces of lepidopteran cellsxe2x80x9d, J. Virol. 72:1085-1091). Sites II and III seem to be close to one another in the three dimensional structure and exposed at the surface of the protein (Gaudin, Y., 1997, xe2x80x9cFolding of rabies virus glycoprotein: epitope acquisition and interaction with endoplasmic reticulum chaperonesxe2x80x9d, J. Virol. 71 :3742-3750). However, at low pH, the G molecule takes on a fusion-inactive conformation in which site II is not accessible to MAbs, whereas sites a and III remain more or less exposed (Gaudin, Y. et al., 1995, xe2x80x9cBiological function of the low-pH, fusion-inactive conformation of rabies virus glycoprotein (G): G is transported in a fusion-inactive state-like conformationxe2x80x9d, J. Virol. 69:5528-5533; Gaudin, Y., et al., 1991, xe2x80x9cReversible conformational changes and fusion activity of rabies virus glycoproteinxe2x80x9d, J. Virol. 65:4853-4859).
Moreover, several regions distributed along the ectodomain are involved in the induction of T helper (Th) cells (MacFarlan, R. et al., 1984, xe2x80x9cT cell responses to cleaved rabies virus glycoprotein and to synthetic peptidesxe2x80x9d, J. Immunol. 133:2748-2752; Wunner, W. et al, 1985, xe2x80x9cLocalization of immunogenic domains on the rabies virus glycoproteinxe2x80x9d, Ann. Inst. Pasteur, 136 E:353-362). Based on these structural and immunological properties, it has been suggested that it the G molecule may contain two immunologically active parts, each potentially able to induce both VNAb and Th cells (Bahloul, C. et al, 1998, xe2x80x9cDNA-based immunization for exploring the enlargement of immunological cross-reactivity against the lyssavirusesxe2x80x9d, Vaccine 16:417-425).
Currently available vaccines predominantly consist of, or are derived from, GT1 viruses, against which they give protection. Many vaccine strains are not effective against GT4, and none are effective against GT2 or GT3. However, the protection elicited against GT4 through 6 depends on the vaccine strain. For example, protection from the European bat lyssaviruses (GT5 and GT6), the isolation of which has become more frequent in recent years, by rabies vaccine strain PM (Pitman-Moore) is not robust. Strain PM induces a weaker protection against EBL1 (GT5) than the protection it provides against strain PV (Pasteur virus).
Because, in part, of the importance of rabies in world health, there is a continuing need to provide safe, effective, fast-acting vaccines and immunogenic compositions to treat and prevent this disease. Many approaches other than use of whole-virus preparations have been proposed and/or pursued to provide an effective, cost-efficient immunogenic composition specific for rabies viruses. For example, as discussed above, subunit vaccines have been developed. Also, vaccines that could generate an immune response to multiple rabies serotypes as well as various other pathogens has been proposed as having some value (European Commission COST/STD-3, 1996, xe2x80x9cAdvantages of combined vaccinesxe2x80x9d, Vaccine 14:693-700). In fact, use of a combined vaccine of diphtheria, tetanus, whole cell pertussis, inactivated poliomyelitis, and rabies has recently been reported (Lang, J. et al., 1997, xe2x80x9cRandomised Feasibility trial of pre-exposure rabies vaccination with DTP-IPV in infantsxe2x80x9d, The Lancet 349:1663-1665). Combined vaccines including rabies have also been used for immunization of dogs (distemper, hepatitis, leptospirosis, and parvo-canine viruses), cats (panleukopenia, calici- and parvo-feline viruses), and cattle (foot and mouth disease virus) (Pastoret, P-P. et al., 1997, xe2x80x9cVaccination against rabiesxe2x80x9d, In Veterinary Vaccinology, Pastoret, P-P. et al., Eds. (Elsevier): 616-628).
Moreover, vaccines produced in tissue culture are expensive to produce despite some attempts to reduce their cost. Consequently DNA vaccines, which are less expensive to produce and offer many advantages, would constitute a valuable alternative. Reports of DNA vaccinations include mouse inoculation with plasmids containing the gene encoding the rabies virus glycoprotein (G). Such inoculation is very potent in inducing humoral and cellular immune responses in association with protection against an intracerebral challenge (see, for example, Lodmell, D. et al., 1998, xe2x80x9cDNA immunization protects nonhuman primates against rabies virusxe2x80x9d, Science Med. 4:949-952; Xiang, Z. et al., 1994, xe2x80x9cVaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virusxe2x80x9d, Virol. 199:132-140; and Lodmell, D. et al., 1998, xe2x80x9cGene gun particle-mediated vaccination with plasmid DNA confers protective immunity against rabies virus infectionxe2x80x9d, Vaccine 16, 115). DNA immunization can also protect nonhuman primates against rabies (Lodmell et al, 1998, supra).
Because administration of plasmid DNA generates humoral and cellular immune responses, including cytotoxic T-Lymphocyte (CTL) production (for review see Donnelly, J. et al., 1997, xe2x80x9cDNA Vaccinesxe2x80x9d, Annu. Rev. Immunol. 15:617-648) and is based on a versatile technology, immunization with plasmid DNA may offer a satisfying prospect for multivalent vaccines. However, the use of a mixture of plasmids or a single plasmid expressing several antigens is believed to induce interference problems at both transcriptional and immunological levels (Thomson, S. et al., 1998, xe2x80x9cDelivery of multiple CD8 cytotoxic cell epitopes by DNA vaccinationxe2x80x9d, J. Immunol. 160: 1717-1723). Therefore, there exists a need to develop and produce multivalent DNA-based vaccines that are effective against rabies and various other diseases; that are safe; and that are cost-efficient to produce and use.
The present invention provides chimeric nucleic acid sequences that encode chimeric polypeptides that induce immunogenic responses in individuals (animals and humans). The nucleic acids of the invention can be expressed to provide chimeric polypeptides that elicit an immune response against rabies and/or rabies-related viruses as well as other pathogenic or otherwise undesirable organisms or polypeptides. Further, the nucleic acids of the invention themselves can elicit at least a portion of the immune response. Thus, the chimeric nucleic acids of the invention can be used to make an immunogenic composition, which can be used to treat an individual.
The present invention also provides a carrier molecule, such as a DNA expression vector, comprising the nucleic acid of the invention, which encodes a chimeric polypeptide. The carrier molecule of the invention can be used as an immunogenic composition, or as part of an immunogenic composition, to elicit the desired immune response. The desired immune response can be a protective response to rabies or rabies-related viruses as well as other organisms or polypeptides. Thus, the carrier molecules of the invention can be used to make an immunogenic composition, which can be used to treat an individual. The carrier molecule can also be used to produce a chimeric polypeptide.
The present invention thus provides a chimeric (fusion) protein that is encoded by the nucleic acid of the invention, or by a nucleic acid sequence present in the carrier molecule of the invention. The chimeric protein can be used to elicit an immunogenic response in an individual. The fusion protein comprises the site III antigenic determinant of a lyssavirus glycoprotein, and can comprise other antigenic sites from one or multiple other polypeptides. Thus, the chimeric polypeptide of the invention can be used to make an immunogenic composition, which can be used to treat an individual.
The present invention further provides immunogenic compositions, including vaccines, that elicit an immunological response in individuals to whom they are administered. The present invention includes immunogenic compositions comprising a polynucleotide sequence that encodes a chimeric (or fusion) polypeptide, or the chimeric polypeptide so encoded, which elicits the immune response. The immunogenic compositions and vaccines of the present invention provide an increased level of immune stimulation and enhanced protection against rabies viruses, and broaden the spectrum of protection against rabies-related viruses, as compared to immunogenic compositions known in the art. The immunogenic compositions also provide multiple immunogenic active sites for induction of an immune response against rabies epitopes as well as epitopes unrelated to rabies.
In view of the above embodiments of the invention, it is evident that the present invention also provides a method of producing a chimeric nucleic acid, a method of producing a carrier molecule, a method of producing a fusion protein, and a method of making an immunogenic composition, such as a vaccine. The immunogenic composition so made can be used to treat (e.g., immunize) individuals.
Included in the invention is the use of the nucleic acid, polypeptide, and/or carrier molecule of the invention to elicit an immune response, such as a protective immune response. Thus, the invention includes the use of a vaccine comprising the polynucleotide, polypeptide, and/or carrier molecule of the invention to treat an individual, either prophylactically or therapeutically. Therefore, the invention includes prophylactic treatment methods, therapeutic treatment methods, and curative treatment methods.